Protein mixtures for wound healing

ABSTRACT

A protein mixture that is useful in the treatment of wounds, where the mixture is isolated from bone or is produced from recombinant proteins and may include two or more of BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7, TGF-β1, TGF-β2, TGF-β3, and FGF-1.

FIELD OF THE INVENTION

The invention relates to use of protein mixtures, comprising a varietyof growth factors, for use in the treatment of wounds.

BACKGROUND OF THE INVENTION

Wound healing is a complex process involving several cell types andgrowth factors for an effective closure. The normal wound healingprocess can be broadly classified into three stages namely theinflammatory, proliferative and maturation phases. The inflammatoryphase lasts 0-2 days and involves an orderly recruitment of cells to thewound area. This is followed by the 2-6 day proliferative phase, inwhich fibroblasts, keratinocytes and other cells in the wound bed beginto actively proliferate to close the wound. The maturation phase followsthe proliferative phase, peaking at 21 days, by which time the wound iscompletely healed by restructuring the initial scar tissue.

A problematic wound does not follow the normal time table for thehealing process as described above. A problematic wound could fail tofollow the normal healing process for any number of reasons, includingnutrition, vascular status, metabolic factors, age, immune status, drugtherapy, neurologic status and psychologic status, among others. Severallocal factors also play an important role in wound healing, includingthe presence of necrotic tissue in the area, infection, foreign bodypresence, degree of desiccation, presence of edema, pressure, friction,shear maceration and dermatitis.

It has been shown from wound fluid composition studies that growthfactors play an important role in all three phases of wound healing. Thecell types that are recruited to the wound area secrete growth factorsthat assist in and promote the wound healing process. Platelets, forexample, are the first cell type to be recruited at the wound site, andinitiate the wound healing process by secreting growth factors (i.e.,platelet derived growth factors, or PDGF) which are chemotactic forother cell types. By so doing, the platelets assist in the recruitmentand proliferation of additional cell types that promote synthesis of newtissue. In addition to the above mentioned functional properties, growthfactors also have the ability to regulate protein synthesis within thecell and control intracellular signaling thus allowing cells tocommunicate with one another.

Since wound healing is a complex process which involves formation ofconnective tissue, and new blood vessels to nourish the site, it isevident that several growth factors come into play. In chronic woundsthere is an increase in collagenase activity and higher levels ofinflammatory cytokines. Additionally, there is an absence of growthfactors in the wound fluid which causes the cells to be mitoticallyincompetent. All of these factors cause impaired wound healing. Some ofthese factors have been studied in the preclinical animal models as wellas in the clinic. Most growth factor studies involving the wound healingprocess involve tests in the 20-25 day range, which appears toadequately model the normal wound healing process. However, it is nowrealized that to get 100% closure of problematic wounds, longer studyperiods such as long as 6 months or more would be advantageous.

The only FDA approved growth factor for wound healing use in the clinicis platelet derived growth factor (PDGF) marketed by Ortho-McNeilPharmacuetical as REGRANEX(r). REGRANEX(r) contains becaplermin, arecombinant human platelet-derived growth factor (rhPDGF-BB) for topicaladministration. Becaplermin is produced by recombinant DNA technology byinsertion of the gene for the B chain of platelet derived growth factor(PDGF) into yeast. Becaplermin has a molecular weight of approximately25 KD and is a homodimer composed of two identical polypeptide chainsthat are bound together by disulfide bonds. REGRANEX(r) is anon-sterile, low bioburden, preserved, sodiumcarboxymethylcellulose-based (CMC) topical gel, containing the activeingredient becaplermin and the inactive ingredients sodium chloride,sodium acetate trihydrate, glacial acetic acid, water for injection, andmethylparaben, propylparaben, and m-cresol as preservatives and 1-lysinehydrochloride as a stabilizer.

Studies of various growth factors in the wound healing process have beenconducted. Some of the findings from these studies are summarized below:

1) PDGF-BB (the growth factor in REGRANEX(r)) is a chemoattractant forneutrophils, monocytes, and fibroblasts. In wound healing applicationsit has been shown to increase extracellular matrix deposition andenhance proliferation of fibroblasts. PDFG is not an angiogen, however.Thus, additional growth factors will be required for the healthymaintenance of neodermis.

2) Fibroblast Growth Factor (FGF) increases capillary density andproliferation of fibroblasts. A topical application in gel form wastested and it was shown that there was no systemic absorption of theprotein (<1% of the dose detected).

3) Transforming growth factor β-2 (TGF β-2) is a growth factor thatenhances proliferation of several cell types both in vitro and in vivoand has been tested in venous ulcer healing and in diabetic foot ulcertrials. In a two arm clinical study a 40% reduction of wound sizecompared to the control wound was observed in 6 weeks when used at 0.5μg/cm2. However, in a 3 arm clinical study when 2.5 μg/cm2 was testedfor comparison against standard XEROFORM(tm) dressing, the results werenot very encouraging.

4) Epidermal growth Factor (EGF) produced by platelets and macrophagesis a mitogen for epithelial cells. This growth factor was first testedin burn patients and the initial results were promising. However, whentested in volunteers there was no difference between growth factortreatments and placebo. This could be due to the fact that EGF is notgood for migration of keratinocytes, but is a good mitotic agent.

5) Keratinocyte Growth Factor-2 (KGF-2) was tested for its ability toincrease ephithelialization. By day 6 the interstices were closed. KGF-2promotes re-epithelialization in young and old animals suggestingindirect mechanisms for neo-granulation tissue formation. Xia Y. D., etal., J. Pathol. (1999) 188: 431-438. There is increased resistance tomechanical stress of healed wounds, hence KGF-2 may be useful for thetreatment of surgical wounds. Jiminez, P. A. & Rampy, M. A., (1999) J.Surg. Res. 81: 238-242.

6) Connective tissue growth factor (CTGF) is a secreted, mitogenic,chemotactic and cell matrix inducing factor encoded by an immediateearly growth responsive gene. Involvement of CTGF in humanatherosclerosis and fibrotic disorders suggests a role in tissueregeneration like wound repair, but also in aberrant deposition ofextracellular matrix. In fact, anti-CTFG antibodies have been used toblock the fibrotic cascade.

Studies on the kinetics of action of various growth factors demonstratedthat some growth factors such as granulocyte-monocyte colony stimulatingfactor (GMCSF) and bovine FGF acted sequentially. It was hypothesizedthat a combination of growth factors would be better than a singlegrowth factor treatment. However, in animal models, a combination ofthese two factors actually slowed the regenerative process and healingnever achieved 100%. Hence, sequential delivery of these factors wasattempted: GMCSF was administered first followed by FGF delivery 25 dayslater. In a single study, no improvement over control could bedemonstrated.

In yet another study combining TGF-β, bFGF (basic FGF) and CTGF it wasfound that TGF-β1, TGF-β2 or TGF-β3 caused skin fibrosis after 3 days ofcontinuous injection but the change was transient and disappeared after7 days of continuous injection. In contrast, irreversible fibrosis wasobserved upon simultaneous injection of TGF-β and bFGF or TGF-β andCTGF, or TGF-β injection for the first 3 days followed by bFGF or CTGFinjection for the next 4 days. These observations suggest that TGF-β1induces skin fibrosis and bFGF or CTGF maintains it in various skinfibrotic disorders.

Another way of obtaining growth factor mixtures considered the use ofplatelet releasate which contains a collection of growth factorsreleased from platelets derived from blood. The advantages of thismaterial are that it is autologous or homologous, and is readilyavailable and presumably contains the required factors in the properratio. To date, although some improvement in the healing process wasobserved initially, by 24 weeks there was no difference between growthfactor and placebo treatments.

It is thus apparent that although several polypeptide growth factorshave shown significant biological activity in pre-clinical wound repairmodels, the only growth factor that has proven to be effective in theclinic is the human recombinant PDGF-BB. This may be due to poordelivery, drug instability or the inability of a single factor toorchestrate the complex process of wound healing. An effective treatmentshould address issues such as angiogenesis, efficient collagendeposition and proper epithelialization to close the wound.

SUMMARY OF THE INVENTION

The invention comprises compositions and methods for improving the wound30 healing process in living animals, including human subjects. Inpreferred embodiments, the invention comprises a mixture of growthfactors which improve the wound healing process. In this context, theterms “excluding,” “exclusion,” or “excluded” refers to the removal ofsubstantially all of an indicated component, to the extent that suchcomponent can be removed from a mixture with inmmunoaffinitychromatography or otherwise not included in the mixture. The term“pharmaceutically acceptable carrier” is used herein in the ordinarysense of the term and includes all known carriers including water.

“BP” is a protein cocktail derived from bone as described in U.S. Pat.Nos. 5,290,763, 5,371,191, and 5,563,124 (each of which is herebyincorporated by reference herein in its entirety). In brief, thecocktail is prepared by guanidine hydrochloride protein extraction ofdemineralized bone particles. The extract solution is filtered, andsubjected to a two step ultrafiltration process. In the firstultrafiltration step an ultrafiltration membrane having a nominalmolecular weight cut off (MWCO) of 100 kD is employed. The retentate isdiscarded and the filtrate is subjected to a second ultrafiltration stepusing an ultrafiltration membrane having a nominal MWCO of about 10 kD.The retentate is then subjected to diafiltration to substitute urea forguanidine. The protein-containing urea solution is then subjected tosequential ion exchange chromatography, first anion exchangechromatography followed by cation exchange chromatography. Theosteoinductive proteins produced by the above process are then subjectedto HPLC with a preparative VYDAC(tm) column at and eluted with shallowincreasing gradient of acetonitrile. One minute fractions of the HPLCcolumn eluate are pooled to make the BP cocktail (fraction number canvary slightly with solvent composition, resin size, volume of productionlot, etc.). One embodiment of the BP cocktail is characterized as shownin FIGS. 1-6. Absolute and relative amounts of the growth factorspresent in the BP cocktail can be varied by collecting differentfractions of the HPLC eluate. In a particularly preferred embodiment,fractions 29-34 are pooled. It is also contemplated that certainproteins may be excluded from the BP mixture without affecting woundhealing activity.

BP was originally discovered as a mixture of proteins known to haveosteogenic activity. However, it contains a plurality of growth factorsand is strongly angiogenic. In particular, BP contains a number of bonemorphogenetic proteins (BMPs), including BMP-2, BMP-3, BMP-4, BMP-5,BMP-6, and BMP-7, as well as TGF-β1, TGF-β2, and TGF-β3. FGF-1 is alsopresent in the mixture. The presence of each of the foregoing proteinswas detected using immunoblot techniques, as depicted FIG. 14. When BPwas tested in an animal model to determine if it would be effective inaiding wound closure, it was surprisingly discovered that BP promoteswound healing, even though it is a markedly different process thanosteogenesis.

The protein compositions of the invention can be advantageously combinedwith traditional wound dressings including primary and secondarydressings, wet-to-dry dressings, absorbent dressings, nonadherentdressings, semipermeable dressings, transparent dressings, hydrocolloiddressings, hydrogels, foam dressings, alginate dressings, surgical tapesand the like as is appropriate for the type of wound being treated.

Compositions according to the present invention may also be combinedwith a variety of other active ingredients, such as aloe vera, arginine,glutamine, zinc, copper, vitamin C, B vitamins and other nutritionalsupplements, antibiotics, antiseptics, antifungals, deodorizers, and thelike. Embodiments of the invention can also be combined with a varietyof anti-inflammatory agents that inhibit the action of proinflammatorycytokines such as interleukin-1, interleukin-6 and tumor necrosisfactor-alpha. Many such inhibitors are well known, such as IL-1Ra,soluble TGF-β receptor, cortocosteroids, and it is believed that morewill be discovered in the future.

In one embodiment, the invention is a composition for the treatment ofwounds comprising the proteins BMP-3 and TGF-β2 in a pharmaceuticallyacceptable carrier. As shown in FIG. 18, BMP-3 is the growth factorpresent in the highest concentration in the BP mixture. TGF-β2 isbelieved to play an important role in wound healing because it promotesthe proliferation of several cell types, which is important, forexample, in the proliferative stage of the wound healing process. Asalready noted, TGF-β2 alone has been the subject of study as a woundhealing agent. Without limitation as to specific mechanisms, it isbelieved that these two growth factors may be significant in the woundhealing activity displayed by BP.

In another embodiment, compositions of the present invention compriseBMP-3, TGF-β2, and one or more of BMP-2, BMP-4, BMP-5, BMP-6, and BMP-7in a pharmaceutically acceptable carrier. BMP-6 is known to induce acascade of events leading to the expression of both BMP-2 and BMP-4,both of which are known to have osteogenic activity. BMP-2 has also beenimplicated in the regulation of kidney tissue regeneration. BMP-7 (alsoknown as OP-1) is currently undergoing preclinical testing as a woundhealing agent.

In still another embodiment, compositions of the present inventioncomprise BMP-3, TGF-β2, one or more of BMP-2, BMP-4, BMP-5, BMP-6, andBMP-7, and one or more of FGF-1, TGF-β1, and TGF-β3. FGF-1 is known tobe an angiogenic growth factor, although its activity is not aspronounced as FGF-2, which has not been detected in BP. TGF-β1 andTGF-β3 are both known to enhance cell proliferation.

The presence of a number of proteins which are believed to have nogrowth factor activity has been detected in BP. Accordingly, theseproteins, including histone proteins, ribosomal proteins, or both, maybe excluded from compositions of the present invention. Alternatively,the composition may comprise the BP mixture isolated as described inU.S. Pat. Nos. 5,290,763, 5,371,191, and 5,563,124 as shown in FIGS. 2and 3 (lanes inside the box pooled to make BP). Histones and ribosomesmay be excluded from the BP by, for example, antibody binding or othertechniques known in the art. Additionally, the composition of matter maycontain one or more of the listed active components supplied as arecombinantly produced protein. Preferably, the components are isolatedfrom a natural source and are at least partially phosphorylated andglycosylated.

In another embodiment, the above compositions are used in wound healingapplications together with a pharmaceutically acceptable carrier. Thepharmaceutically acceptable carrier includes dressings such ashydrocolloid dressings, hydrogels, foam dressings, and alginatedressings. Additional active ingredients may include arginine,glutamine, zinc, copper, vitamin C, vitamin B1, vitamin B2, vitamin B3,vitamin B6, vitamin B12, and folate or growth factors such as epidermalgrowth factor, platelet derived growth factor, insulin-like growthfactor, keratinocyte growth factor, vascular endothelial growth factor,transforming growth factor alpha, nerve growth factor, connective tissuegrowth factor and granulocyte-monocyte colony stimulating factor.Inflammation inhibitor, such as interleukin-1 inhibitor, interleukin-6inhibitor and tumor necrosis factor-alpha inhibitor may also be added tothe composition. Of course, pain relief agents, disinfectants,antibiotics and other active ingredients suitable for particular woundapplications may also be added thereto.

DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates an SDS-PAGE of a protein mixture according to thepresent invention, both in reduced and nonreduced forms.

FIG. 2 is an SDS-PAGE gel of HPLC fractions 27-36 of a protein mixtureaccording to an embodiment of the present invention.

FIG. 3 is an SDS-PAGE gel with identified bands indicated according tothe legend of FIG. 4.

FIG. 4 is an SDS-PAGE gel of a protein mixture according to anembodiment of the present invention with identified bands indicated, asprovided in the legend.

FIG. 5 is two dimensional (2-D) SDS-PAGE gel of a protein mixtureaccording to an embodiment of the present invention with internalstandards indicated by arrows.

FIG. 6 is a 2-D SDS-PAGE gel of a protein mixture according to anembodiment of the present invention with circled proteins identified asin the legend.

FIGS. 7A-O are mass spectrometer results for tryptic fragments from onedimensional (1-D) gels of a protein mixture according to an embodimentof the present invention.

FIG. 8 is a 2-D gel Western blot of a protein mixture according to anembodiment of the present invention labeled with anti-phosphotyrosineantibody.

FIGS. 9A-D are 2-D gel Western blots of a protein mixture according toan embodiment of the present invention, labeled with indicatedantibodies. FIG. 9A indicates the presence of BMP-3 and BMP-2. FIG. 9Bindicates the presence of BMP-3 and BMP-7. FIG. 9C indicates thepresence of BMP-7 and BMP-2, and FIG. 9D indicates the presence of BMP-3and TGF-β1.

FIG. 10 is a PAS (periodic acid schiff) stained SDS-PAGE gel of HPLCfractions of a protein mixture according to an embodiment of the presentinvention.

FIG. 11 is an anti-BMP-7 stained SDS-PAGE gel of a PNGase F treatedprotein mixture according to an embodiment of the present invention.

FIG. 12 is an anti-BMP-2 stained SDS-PAGE gel of a PNGase F treatedprotein mixture according to an embodiment of the present invention.

FIGS. 13A-B are bar charts showing explant mass of glycosylatedcomponents in a protein mixture according to an embodiment of thepresent invention (FIG. 13A) and ALP score (FIG. 13B) of the samecomponents.

FIG. 14 is a chart showing antibody listing and reactivity.

FIGS. 15A-B together comprise a chart showing tryptic fragmentsequencing data for components of a protein mixture according to anembodiment of the present invention.

FIGS. 16A-F together comprise a chart showing tryptic fragment massspectrometry data for components of a protein mixture according to anembodiment of the present invention.

FIGS. 17A-B are an SDS-gel (FIG. 17B) and a scanning densitometer scan(FIG. 17A) of the same gel for a protein mixture according to anembodiment of the present invention.

FIG. 18 is a chart illustrating the relative mass, from scanningdensitometer quantification, of protein components in a protein mixtureaccording to an embodiment of the present invention.

FIGS. 19A-D together comprise a chart showing mass spectrometry data ofvarious protein fragments from 2D gels of a protein mixture according toan embodiment of the present invention.

DETAILED DESCRIPTION OF THE INVENTION EXAMPLE 1 BP IN SINGLE DOSEAPPLICATION TO NUDE MICE

A single dose application of BP to full thickness wounds in nude micecovered with human meshed split thickness skin grafts has been found toheal the wound completely and faster than wounds not receiving thegrowth factor mixture. Although the specific manner in which the growthfactors in BP affect the wound healing process is not fully understood,it is hypothesized that the synergistic action of the multiple growthfactors present in BP helps the wounds recover better than those incontrol animals that have received the carrier alone.

Full thickness wounds were created in nude mice such that the wound areacomprised about 20% of the total body surface. BP was prepared as inU.S. Pat. Nos. 5,290,763, 5,371,191, and 5,563,124, and applied to thewound in a povidone carrier. The wound was then covered with humanmeshed split thickness skin grafts. The control group of animalsreceived only the povidone carrier. The graft sites were dressed andclosed with band-aids to keep the dressing securely in place. The firstdressing changes were carried out on day 5 post operative and everythird day thereafter. The basic protocol is also described in “Clinicaland Experimental Approaches to Dermal and Epidermal Repair: Normal andChronic Wounds,” pp. 429-442 (1991) Wiley-Liss, Inc. and Cooper M. L.,et al., The Effects of Epidermal Growth factor and basic FibroblastGrowth factor on Epithelialization of Meshed Skin Graft Interstices,Prog. Clin. Biol. Res. (1991) 365: 429-42. Such protocols are known topersons of skill in the art.

The results were strongly encouraging. Single application of twoconcentrations (either 100 μg/wound site or 200 μg/wound site) of growthfactor were tested. There was no difference either in the rate or degreeof wound healing between the two groups. However, there was a markeddifference between the group of animals that received the growth factortreatment and the control animals that did not receive the growthfactor. By day 11 POD (post operative day), a 95% wound closure wasobserved in the animals that received the growth factor whereas thecontrol animals showed only a 74% closure. By day 14 POD all growthfactor treated animals had a 100% closure while the control animals hadonly a 85% closure as of day 20 POD.

The thickness of the epithelial layer in BP treated wounds wassignificantly higher in BP treated animals compared to the controlanimals, as shown in Table 1. The data represents the thickness ofneodermis in mm measured on day 11 for the BP treated animals and day 16for the control animals such that measurements are made at equivalentextents of healing. Histological analysis revealed that the wounds wereclosed by the human cells from the grafted material and there wascollagen deposition in the closed wounds as revealed by involucrin andcollagen type 1 immuno histological staining (data not shown). Thecapillary density in the wound bed following BP treatment was alsosignificantly higher at the time of wound closure compared to untreatedcontrols, as shown in Table 1. Further, in the animals treated with thelower BP dosage, there was a significant increase in the smooth musclecell (SMC) count in the BP treated wounds as compared to the controls,as also seen in Table 1. TABLE 1 Wound Thickness, Capillary Count andSMC Count for BP and Control Treated Wounds. Treatment 100 μg BP 200 μgBP Control (n = 5) (n = 5) (n = 10) Epithelial Thickness 1.60 ± 0.121.55 ± 0.09  1.1 ± 0.25 (mm) (P < 0.001) (P < 0.001) Capillary/Field 37± 6  35 ± 7  25 ± 5.9 (P < 0.01) (P < 0.01) SMC counts/Field  53 ± 3.546.8 ± 4.4  46 ± 5.8 (P < 0.001) (P < 0.05)

In summary, a single dose application of BP was effective in reducingthe healing time of full thickness wound in nude mice grafted with humanmeshed split thickness skin. Additionally, the thickness of theneodermis and the density of the capillaries in the treated wounds weresignificantly higher compared to the control group of animals. Incontrast, bFGF, also an angiogenic growth factor, was shown to have adeleterious effect on epithelialization when tested in a similar animalmodel. (Cooper, M. L. et al., 1991; Clinical and experimental approachesto dermal and epidermal repair: normal and chronic wounds, pp 429-442;Weilly-Liss, Inc.).

EXAMPLE 2 BP IN HYDROGEL

A small number of animals (n=3) were treated with BP solubilized in ahydrogel (carboxy-methyl cellulose) in the same animal model asdescribed above. In this study, it was observed that the wounds (n=2)treated with BP in the hydrogel showed initiation of epithelializationas early as 5 days post operation compared to the wounds treated with BPsolubilized in 1% povidone which showed initiation of epithelializationonly at 8 days post operation (data not shown). In both instances, thecontrol animals that received the carrier alone did not show initiationof epithelialization until POD 8. Detailed histology is being carriedout on the tissue samples to determine the thickness of the neodermisand the degree of angiogenesis in the wounds treated with the hydrogelformulation. However, wound closure data is presented in Table 2, below.TABLE 2 Percent Wound Closure for BP and Control Treated Wounds. PercentWound Closure (%) Animal POD POD POD POD # 5 8 11 14 *Control (no BP) 10 50 70 70 Control (hydrogel, no BP, no salts) 2 25 70 70 100 BP &hydrogel, no salts 3 0 70 90 100 BP & hydrogel, no salts 4 25 80 90 90BP & hydrogel, salts (some 5 0 80 90 100 precipitate formed, probablydue to buffering salts)*The control animal had very thin and fragile skin at the time of biopsycompared to the animals which received BP.

In summary, the results were very promising although preliminary,showing quicker wound closure in BP treated than control animals. Thus,more extensive experiments were undertaken to confirm the results, asdescribed below.

EXAMPLE 3 COMPARATIVE STUDY BETWEEN REGRENEX(r) AND BP

REGRANEX(r) (PDGF-BB), the only approved growth factor product in themarket for treating diabetic foot ulcers, showed complete healing in 50%of the patient population compared to the 35% placebo gel treatment thatdemonstrated complete healing after repeat application for about 20weeks in diabetic patients (see REGRANEX(r) U.S. full prescribinginformation—package insert). Hence, a comparison of REGRANEX(r) (tm)versus BP was undertaken in a study similar to that described above. Theresults are presented in Tables 3 and 4. TABLE 3 BP, Hydrogel (HG) andRegranex ® Treated Wounds and Percent Wound Closure (%), EpithelialThickness (mm) and Degree of Angiogenesis (# Estimated Capillaries per20x Field). Angio. Epi. (# est. Thick. cap/hpf Percent (%) Wound Closure(μm) 20x) Animal # Treatment Group POD 5 POD 8 POD 11 POD 14 POD 14 POD14  1 BP 10 25 85 100 17.5 28  2 BP 10  3 BP 15  4 BP 10  5 BP 10 30 8580 7.5 16  6 BP 10  7 BP 10 10  8 BP 10 30 85 100 11.5 26  9 BP 30 50 85100 16 21 10 BP 30 50 85 100 12 20 11 BP 20 45 85 100 18 18 12 BP 10 1585 90 6 20 13 BP 10 20 95 100 5.5 23 14 BP 15 25 90 100 10 32 15 BP 5 5090 95 14 25 n 15 11 10 10 10 10 mean 13.67 31.82 87.00 96.50 11.80 22.9SD 7.43 14.71 3.50 6.69 4.58 4.88 SEM 0.54 0.46 0.04 0.07 0.39 16 HG 1535 75 55 12.5 28 17 HG 10 60 70 95 10.5 5 18 HG 5 25 60 95 9 34 19 HG 1030 70 90 17.5 8 20 HG 20 40 80 95 17.5 20 21 HG 10 10 80 95 13 15 22 HG30 80 70 90 10 23 HG 10 80 80 90 20 10 24 HG 15 40 70 90 18 15 25 HG 2035 70 90 10.5 16 26 HG 10 10 70 90 12.5 20 27 HG 10 35 70 90 8 32 28 HG10 55 29 HG 5 40 30 HG 15 40 70 n 15 15 13 12 12 11 mean 13.00 41.0071.92 88.75 13.25 18.455 SD 6.49 20.72 5.60 10.90 4.01 9.55 SEM 0.500.51 0.08 0.12 0.30 31 Regranex 20 30 55 75 16 32 Regranex 15 80 13 33Regranex 20 80 100 100 8.5 4 34 Regranex 15 50 90 100 10 35 Regranex 4075 6 36 Regranex 15 70 90 100 7.5 10 37 Regranex 15 70 90 18 38 Regranex10 80 39 Regranex 40 80 40 Regranex 15 50 80 90 15 13 41 Regranex 15 1042 Regranex 5 50 100 100 16 21 43 Regranex 40 70 100 100 22.5 10 44Regranex 5 40 80 100 16.5 6 45 Regranex n 14 14 9 8 9 9 mean 19.29 59.6487.22 95.63 14.44 10.375 SD 12.07 21.88 14.39 9.04 4.88 5.4 SEM 0.630.37 0.16 0.09 0.34

The percent closure results can be summarized as follows: TABLE 4Summary POD's BP (mean) HG (mean) REG (mean) wound closure (%) 0 0.000.00 0.00 5 13.67 13.00 19.29 8 31.82 41.33 59.64 11 87.00 71.92 87.2214 96.25 89.17 95.63 epithelial thickness 14 11.8 13.25 14.44 (mm)angiogenesis (#/filed) 14 22.9 18.45 10.38

Thus, the BP treatment is as good as REGRENEX(tm) in closing woundsalthough slightly slower healing rates are initially observed. BPtreatment also shows slightly less thickening of the epithelium andshows considerably improved angiogenesis in the wound area.

EXAMPLE 4 FUTURE APPLICATIONS

Because BP has shown promise as a wound healing agent, it will next betested in applications where wound healing is known to be deficient.Experiments similar to those described above will be performed withdiabetic animals to test the healing of full and partial thicknesswounds. The response of venous stasis ulcers and diabetic ulcers to BPwill also be tested.

In preliminary experiments, Male Sprague Dawley rats weighing greaterthan 325 g were rendered diabetic by treatment with streptozotocin andthe hyperglycemia was confirmed by glucometry. Four full thicknessincisional wounds were introduced on the dorsal surface of each animalperpendicular to the longitudinal axis. The wounds were closed with silksutures and the growth factor or the placebo applied into the wound gapor on top of the incision after closure. The application was done at twotime points: 1) on day 0, which is on the day of introducing the wound(surgery) and a second application 2) on day 3 following theintroduction of the wound. The incisional strength was measured on day 7and day 10 after surgery. The data is given in Table 5 and is veryencouraging that the BP treatment will be particularly useful intreating a variety of diabetic ulcers, or other wounds characterized bydelayed and/or poor healing. TABLE 5 Tensile Strength of Wounds inDiabetic Rats Tensile Strength (kg/mm) ± sem Control BP Day 7 3.6 ± .14.2 ± .7 Day 10 5.2 ± .7 9.1 ± .8

EXAMPLE 5 FURTHER CHARACTERIZATION OF BP

The BP has been partially characterized as follows: high performanceliquid chromatography (“HPLC”) fractions have been denatured, reducedwith DTT, and separated by sodium dodecyl sulfate polyacrylamide gelelectrophoresis (SDS-PAGE). One minute HPLC fractions from 27 to 36minutes are shown in FIG. 2. Size standards (ST) of 14, 21, 31, 45, 68and 97 kDa were obtained as Low Range size standards from BIORAD(tm) andare shown at either end of the coomassie blue stained gel. In the usualprotocol, HPLC fractions 29 through 34 are pooled to produce BP (seeboxes, FIGS. 2 and 3), as shown in a similarly prepared SDS-PAGE gel inFIG. 17B.

The various components of the BP were characterized by mass spectrometryand amino acid sequencing of tryptic fragments where there weresufficient levels of protein for analysis. The major bands in the ID gel(as numerically identified in FIG. 3) were excised, eluted, subjected totryptic digestion and the fragments were HPLC purified and sequenced.The sequence data was compared against known sequences, and the bestmatches are shown in FIGS. 15A-B. These identifications are somewhattentative in that only portions of the entire proteins have beensequenced and, in some cases, there is variation between the human andbovine analogs for a given protein.

The same tryptic protein fragments were analyzed by mass spectrometryand the mass spectrograms are shown in FIGS. 7A-O. The tabulated resultsand homologies are shown in FIGS. 16A-F which provides identificationinformation for the bands identified in FIGS. 3-4. As above, assignmentof spot identity may be tentative based on species differences and posttranslational modifications. A summary of all protein identificationsfrom ID gels is shown in FIG. 4.

The identified protein components of BP, as described in FIGS. 15A-B,16A-F and 19A-D, were quantified as shown in FIGS. 17A and 17B. FIG. 17Bis a stained SDS-PAGE gel of BP and FIG. 17A represents a scanningdensitometer trace of the same gel. The identified proteins were labeledand quantified by measuring the area under the curve. These results arepresented in FIG. 18 as a percentage of the total peak area.

Thus, there are 11 major bands in the BP SDS-PAGE gel representing about60% of the protein in BP. The identified proteins fall roughly intothree categories: the ribosomal proteins, the histones and growthfactors, including bone morphogenic factors (BMPs). It is expected thatthe ribosomal proteins and histone proteins may be removed from the BPwithout loss of activity, since these proteins are known to have nogrowth factor activity. Upon this separation, the specific activity isexpected to increase correspondingly.

Experiments are planned to confirm the hypothesis that the histone andribosomal proteins may be removed from the BP with no resulting loss, oreven an increase, in specific activity. Histones will be removed fromthe BP cocktail by immunoaffinity chromatography using either specifichistone protein antibodies or a pan-histone antibody. The histonedepleted BP (BP-H) will be tested as described above for wound healingand/or osteogenic activity. Similarly, the known ribosomal proteins willbe stripped and the remaining mixture (BP-R) tested. An SDS-PAGE gel ofBP was also analyzed by Western immunoblot with a series of antibodies,as listed in FIG. 14. Visualization of antibody reactivity was by horseradish peroxidase conjugated to a second antibody and using achemiluminescent substrate. Further, TGF-β1 was quantified usingcommercially pure TGF-β1 as a standard and was determined to representless than 1% of the BP protein The antibody analysis indicated that eachof the proteins listed in FIG. 14 is present in BP.

The BP was further characterized by 2-D gel electrophoresis, as shown inFIGS. 5-6. The proteins are separated in horizontal direction accordingto charge (pI) and in the vertical direction by size as described intwo-dimensional electrophoresis adapted for resolution of basic proteinswas performed according to the method of O'Farrell et al. (O'Farrell, P.Z., Goodman, H. M. and O'Farrell, P. H., Cell, 12: 1133-1142, 1977) bythe Kendrick Laboratory (Madison, Wis.). Two-dimensional gelelectrophoresis techniques are known to those of skill in the art.Nonequilibrium pH gradient electrophoresis (“NEPHGE”) using 1.5% pH3.5-10, and 0.25% pH 9-11 ampholines (Amersham Pharmacia Biotech,Piscataway, N.J.) was carried out at 200 V for 12 hrs. Purifiedtropomyosin (lower spot, 33,000 KDa, pI 5.2), and purified lysozyme(14,000 KDa, pI 10.5-11) (Merck Index) were added to the samples asinternal pI markers and are marked with arrows.

After equilibration for 10 min in buffer “0” (10% glycerol, 50 mMdithiothreitol, 2.3% SDS and 0.0625 M tris, pH 6.8) the tube gel wassealed to the top of a stacking gel which is on top of a 12.5%acrylamide slab gel (0.75 mm thick). SDS slab gel electrophoresis wascarried out for about 4 hrs at 12.5 mA/gel.

After slab gel electrophoresis two of the gels were coomassie bluestained and the other two were transferred to transfer buffer (12.5 mMTris, pH 8.8, 86 mM Glycine, 10% MeoH) transblotted onto PVDF paperovernight at 200 mA and approximately 100 volts/two gels. The followingproteins (Sigma Chemical Co., St. Louis, Mo.) were added as molecularweight standards to the agarose which sealed the tube gel to the slabgel: myosin (220,000 KDa), phosphorylase A (94,000 KDa), catalase(60,000 KDa), actin (43,000 KDa), carbonic anhydrase (29,000 KDa) andlysozyme (14,000 KDa). FIG. 5 shows the stained 2-D gel with sizestandards indicated on the left. Tropomyosin (left arrow) and lysozyme(right arrow) are also indicated.

The same gel is shown in FIG. 6 with several identified proteinsindicated by numbered circles. The proteins were identified by massspectrometry and amino acid sequencing of tryptic peptides, as describedabove. The identity of each of the labeled circles is provided in thelegend of FIG. 6 and the data identifying the various protein spots ispresented in FIGS. 19A-D. Because several of the proteins migrated atmore than one size (e.g., BMP-3 migrating as 6 bands) investigationswere undertaken to investigate the extent of post-translationmodification of the BP components. Phosphorylation was measured byanti-phosphotyrosine immunoblot and by phosphatase studies. FIG. 8 showsa 2-D gel, electroblotted onto filter paper and probed with aphosphotyrosine mouse monoclonal antibody by SIGMA (#A-5964). Severalproteins were thus shown to be phosphorylated at one or more tyrosineresidues.

Similar 2-D electroblots were probed with BP component specificantibodies, as shown in FIGS. 9A-D. The filters were probed with BMP-2,BMP-3 (FIG. 9A), BMP-3, BMP-7 (FIG. 9B), BMP-7, BMP-2 (FIG. 9C), andBMP-3 and TGF-β1 (FIG. 9D). Each shows the characteristic, single-sizeband migrating at varying pI, as is typical of a protein existing invarious phosphorylation states.

For the phosphatase studies, BP in 10 mM HCl was incubated overnight at37° C. with 0.4 units of acid phosphatase (AcP). Treated and untreatedsamples were added to lyophilized discs of type I collagen and evaluatedside by side in the subcutaneous implant rat bioassay, as previouslydescribed in U.S. Pat. Nos. 5,290,763, 5,563,124 and 5,371,191. Briefly,10 (g of BP in solution was added to lyophilized collagen discs and thediscs implanted subcutaneously in the chest of a rat. The discs werethen recovered from the rat at 2 weeks for the alkaline phosphotase(“ALP”—a marker for bone and cartilage producing cells) assay or at 3weeks for histological analysis. For ALP analysis of the samples, theexplants were homogenized and levels of ALP activity measured using acommercial kit. For histology, thin sections of the explant were cutwith a microtome, and the sections stained and analyzed for bone andcartilage formation.

Both native- and phosphatase-treated BP samples were assayed formorphogenic activity by mass of the subcutaneous implant (explant mass)and ALP score. The results showed that AcP treatment reduced the explantmass and ALP score from 100% to about 60%. Thus, phosphorylation isimportant for BP activity. The BP was also analyzed for glycosylation.FIG. 10 shows an SDS-PAGE gel stained with periodic acid schiff (PAS)—anon-specific carbohydrate stain, indicating that several of the BPcomponents are glycosylated (starred protein identified as BMP-3). FIGS.11-12 show immunodetection of two specific proteins (BMP-7, FIG. 11 andBMP-2, FIG. 12) treated with increasing levels of PNGase F(Peptide-N-Glycosidase F). Both BMP-2 and BMP-7 show some degree ofglycoslyation in BP, but appear to have some level of protein resistantto PNGase F as well (plus signs indicate increasing levels of enzyme).Functional activity of PNGase F and sialadase treated samples wereassayed by explant mass and by ALP score, as shown in FIG. 13A and 13Bwhich shows that glycosylation is required for full activity.

In summary, BMPs 2, 3 and 7 are modified by phosphorylation andglycosylation. These post-translation modifications affect proteinmorphogenic activity, 33% and 50% repectively, and care must be taken inpreparing BP not to degrade these functional derivatives.

1.-24. (canceled)
 25. A method of promoting the healing of a skin woundcomprising applying to the skin wound an effective skin wound healingamount of a composition comprising: a mixture of osteogenic proteins inwhich BMP-3 is the growth factor present in highest concentration; and apharmaceutically acceptable carrier.
 26. The method of claim 25, whereinat least one of the mixture of osteogenic proteins is derived from bone.27. The method of claim 26, wherein the bone comprises bovine bone. 28.The method of claim 26, wherein the bone comprises demineralized bone.29. The method of claim 25, wherein the composition further comprises atleast one growth factor selected from the group consisting of FGF-1,TGF-β1, TGF-β2 and TGF-β3.
 30. The method of claim 25, wherein thecomposition further comprises TGF-β2.
 31. The method of claim 30,wherein the composition further comprises BMP-6.
 32. The method of claim25, wherein the composition further comprises BMP-6.
 33. The method ofclaim 25, wherein the composition further comprises BMP-2.
 34. Themethod of claim 25, wherein the composition further comprises at leastone growth factor selected from the group consisting of BMP-2, BMP-4,BMP-5, BMP-6, and BMP-7.
 35. A method of promoting skin wound healingcomprising applying to the skin wound a composition comprising a mixtureof growth factors comprising BMP-2, BMP-3, BMP-7, and TGF-β1 in apharmaceutically acceptable carrier.
 36. The method of claim 35, whereinat least one of the mixture of growth factors is derived from bone. 37.The method of claim 36, wherein the bone comprises bovine bone.
 38. Themethod of claim 36, wherein the bone comprises demineralized bone. 39.The method of claim 35, wherein the composition further comprises atleast one growth factor selected from the group consisting of FGF-1,TGF-β2 and TGF-β3.
 40. The method of claim 35, wherein the compositionfurther comprises FGF-1.
 41. The method of claim 35, wherein thecomposition further comprises BMP-4.
 42. The method of claim 35, whereinthe composition further comprises BMP-5.
 43. The method of claim 35,wherein the composition further comprises BMP-6.
 44. A method ofpromoting skin wound healing comprising applying to the skin wound acomposition comprising a mixture of growth factors comprising BMP-2,BMP-3, BMP-4, BMP-5, BMP-6, BMP-7, TGF-β1, TGF-β2, TGF-β3 and FGF-1 in apharmaceutically acceptable carrier.